Cephem compounds having at the 3-position a (1,4-methylene-1-piperidinio)methyl group or a (1-quinuclidinio)methyl group

ABSTRACT

Described herein is a cephem derivative represented by the general formula: ##STR1## wherein n stands for 1 or 2, Y stands for CH or nitrogen atoms, R 1  represents a lower hydrocarbon group or a carboxyl-substituted, a carbamoyl-substituted, or a cyclopropyl-substituted lower alkyl group, and R 2  denotes hydroxyl group, a lower alkyl group, a hydroxy-substituted lower alkyl group, or carbamoyl group. The derivative is useful as an antibacterial agent. Also described herein are processes for the production of the derivative, antibacterial composition, intermediate of the derivative and process for the production thereof.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel cephem derivatives useful asantibacterial agents, process for the production thereof, antibacterialcomposition, intermediates thereof and process therefor.

2. Description of the Prior Art

A number of compounds have heretofore been known which contain asubstituted thiadiazolylacetamido group or substitutedthiazolylacetamido group at the 7-position of the cephem skeleton. Forexample, the compounds may be disclosed in the following publications:Japanese Patent Application Laid-open Nos. 11600/1980, 105689/1980,24389/1982, 81493/1982, 4789/1983, 41887/1983, 59992/1983, 149296/1981,102293/1977, 116492/1977, 125190/1977, 154786/1979, 192394/1982,219292/1984, 97982/1985, 197693/1985, 231683/85, etc.

Particularly,7β-[(Z)-2-(2-aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-(1-quinuclidinio)methyl-3-cephem-4-carboxylateis described in Japanese Patent Application Laid-open Nos. 219292/1984,197693/1985, and 231683/1985. However, this compound can not bepractically used from a clinical point of view, because its acute toxicvalue [LD₅₀ (mouse, intravenous injection)] amounts to about 100 mg/kgor less, and hence, the compound is very toxic.

SUMMARY OF THE INVENTION

The present inventors have found that cephem derivatives, each of whichhas the hereinunder-described group at the 3-position of the cephemskeleton, have excellent antibacterial activities, leading to completionof this invention: ##STR2## wherein n stands for 1 or 2 and R₂ denoteshydroxyl group, a lower alkyl, a hydroxy-substituted lower alkyl group,or carbamoyl group.

An object of this invention is therefore to provide novel compoundsuseful as antibacterial agents, their production process and their useas an antibacterial composition.

Further object of this invention is to provide novel intermediates ofthe above cephem derivatives and the process for the production thereof.

DESCRIPTION OF THE INVENTION

The compounds of this invention are defined as follows:

A cephem derivative represented by the general formula: ##STR3## whereinn stands for 1 or 2, Y stands for CH or nitrogen atom, R₁ represents alower hydrocarbon group or a carboxyl-substituted, acarbamoyl-substituted, or a cyclopropyl-substituted lower alkyl group,and R₂ denotes hydroxyl group, a lower alkyl group, ahydroxy-substituted lower alkyl group, or carbamoyl group, or apharmaceutically acceptable salt thereof.

As the lower hydrocarbon group represented by R₁ in the general formula(I), there may be mentioned lower alkyl groups such as methyl, ethyl,n-propyl, i-propyl, n-butyl, t-butyl and sec-butyl; lower alkenyl groupssuch as vinyl and allyl; lower alkynyl groups such as propargyl; and thelike. Examples of the carboxy-substituted lower alkyl group representedby R₁ may include carboxymethyl, 2-carboxy-ethyl, 3-carboxypropyl,1-carboxyethyl, 1-carboxy-1-methylethyl, and the like. Exemplarycarbamoyl-substituted lower alkyl groups represented by R₁ may includecarbamoylmethyl, 2-carbamoylethyl, 3-carbamoylpropyl,1-carbamoyl-1-methylethyl, 1-carbamoylethyl and the like. Exemplarycyclopropyl-substituted lower alkyl groups represented by R₁ may includecyclopropylmethyl, 2-cyclopropylethyl, and the like.

As exemplary hydroxy-substituted lower alkyl groups represented by R₂,there may be mentioned hydroxy-methyl, 2-hydroxyethyl, 1-hydroxyethyl,3-hydroxypropyl and the like. Exemplary lower alkyl groups representedby R₂ may include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl,and the like.

As non-toxic salts of the compounds of the general formula (I), may bementioned their pharmaceutically-acceptable salts, for example, alkalimetal salts such as sodium salts and potassium salts; alkaline earthmetal salts such as calcium salts and magnesium salts; inorganic acidsalts such as hydrochlorides, hydrobromides, hydroiodides, sulfates,carbonates and bicarbonates; organic carboxylates such as maleates,lactates and tartrates; organic sulfonates such as methanesulfonates,benzenesulfonates and toluenesulfonates; amino acid salts such asarginine salts, lysine salts, serine salts, aspartates and glutamates;amine salts such as trimethylamine salts, triethylamine salts, pyridinesalts, procaine salts, picoline salts, dicyclohexylamine salts,N,N'-dibenzylethylenediamine salts, N-methylglucamine salts,diethanolamine salts, triethanolamine salts,tris(hydroxymethylamino)methane salts and phenethylbenzylamine salts;and the like.

Each of the compounds of the general formula (I), which pertains to thepresent invention, has its syn-isomer (Z) and anti-isomer (E) withrespect to its configuration at the following moiety: ##STR4## Althoughboth isomers are included in the present invention, the syn-isomers aredesired owing to their anti-bacterial activities.

The compounds of this invention can be produced by the followingprocesses.

I: First Process for Production:

The compounds of the general formula (I) and their pharmaceuticallyacceptable salts can be obtained by reacting a compound represented bythe general formula: ##STR5## wherein Y and R₁ have the same meanings asdefined above and X represents a halogen atom, or a compound wherein theamino group and/or carboxyl group is/are protected by a protectivegroup, or a salt thereof, with a compound represented by the generalformula: ##STR6## wherein n and R₂ have the same meanings as definedabove, or with a salt thereof, followed by optionally removing theprotective group.

As halogen atoms represented by X in the above general formula (II),there may be mentioned iodine atom, bromine atom and chlorine atom. Ofthese, iodine atom and bromine atom are particularly desired.

The above reaction may be carried out at a reaction temperature of -10°C. to 60° C. or preferably 0° C. to 40° C. As a reaction solvent, ananhydrous organic solvent is desired. As usable organic solvents, theremay be mentioned lower alkylnitriles such as acetonitrile andpropionitrile; halogenated lower alkyls such as chloromethane, methylenechloride and chloroform; ethers such as tetrahydrofuran and dioxane;amides such as N,N-dimethylformamide; esters such as ethyl acetate;ketones such as acetone; and hydrocarbons such as benzene; as well asmixed solvents thereof.

As the salts of the compounds of the general formulae (II) and (III) andthe protective groups for the amino group and carboxyl group in thecompounds of the general formula (II), those employed routinely may alsobe used so long as they do not impair the reaction.

For example, the formyl group, acetyl group, chloroacetyl group,dichloroacetyl group, t-butoxycarbonyl group, benzyloxycarbonyl group,trityl group, p-methoxybenzyl group, diphenylmethyl group and the likemay be used as protective groups for amino group; and p-methoxybenzylgroup, p-nitrobenzyl group, t-butyl group, methyl group,2,2,2-trichloroethyl group, diphenylmethyl group, pivaloyloxymethylgroup and the like as protective groups for carboxyl group. Further, useof a silylating agent such as bis(trimethylsilyl)acetamide,N-methyl-N-(trimethylsilyl)acetamide orN-methyl-N-trimethylsilyltrifluoroacetamide and the like is convenientbecause such a silylating agent can protect both amino and carboxylgroups at the same time.

As salts of the compounds of the general formulae (II) and (III),suitable selection may be made from their salts such as their alkalimetal salts such as sodium salts and potassium salts; alkaline earthmetal salts such as calcium salts and magnesium salts; ammonium salts;inorganic acid salts such as hydrochlorides, hydrobromides, sulfates,carbonates, hydroiodides and bicarbonates; organic carboxylates such asacetates, maleates, lactates and tartrates; organic sulfonates such asmethanesulfonates, benzenesulfonates and toluenesulfonates; amine saltssuch as trimethylamine salts, triethylamine salts, pyridine salts,procaine salts, picoline salts, dicylohexylamine salts,N,N'-dibenzylethylenediamine salts, N-methylglucamine salts,diethanolamine salts, triethanolamine salts,tris(hydroxymethylamino)methane salts and phenethylbenzylamine salts;amino acid salts such as arginine salts, aspartates, lysine salts,glutamates and serine salts; and the like.

The removal of the protective group can be carried out by anyconventional processes such as hydrolysis, reduction, and the like,depending on the types of the protective groups used.

II: Second Process for Production:

The compounds of the general formula (I) and their pharmaceuticallyacceptable salts can also be obtained by reacting a compound representedby the general formula: ##STR7## wherein n and R₂ have the same meaningsas defined above, or a compound wherein the group --COO⁻ has beenprotected by a protective group, or a salt thereof, with a compoundrepresented by the general formula: ##STR8## wherein Y and R₁ have thesame meanings as defined above, or a compound wherein the amino grouphas been protected by a protective group, or a reactive derivative atthe carboxyl group thereof, or a salt thereof, and followed byoptionally removing the protective group.

The process may be carried out in accordance with any conventionalN-acylating reaction conditions. For example, the reaction may beperformed in an inert solvent at a temperature of -50° C. to 50° C.,preferably -20° C. to 30° C. in the presence or absence of a base.Examples of the inert solvent include acetone, tetrahydrofuran,N,N-dimethylformamide, N,N-dimethylacetamide, dioxane, dichloromethane,chloroform, benzene, toluene, acetonitrile, or the mixed solventsthereof. Examples of the base include N,N-dimethylaniline,triethylamine, pyridine, N-methylmorpholine and the like.

In the case where the carboxylic acids (--COOH) represented by thegeneral formula (V) are used in the process according to the presentinvention, the reaction is preferably carried out in the presence of acondensation agent such as N,N'-dicyclohexylcarbodiimide,N,N'-diethylcarbodiimide, N-cyclohexyl-N'-morpholinoethylcarbodiimide,trialkyl phosphite, ethyl polyphosphate, p-toluenesulfonic acid chlorideand the like. Furthermore, in the case where such a reactive derivativeat the carboxyl group in the general formula (V) is used, an example ofthe reactive derivative includes acid halides such as acid chloride,acid bromide and the like; symmetrical acid anhydrides; mixed acidanhydrides with carboxylic acid such as ethyl chlorocarbonate,trimethylacetic acid, thioacetic acid, diphenylacetic acid and the like;active esters with 2-mercaptopyridine, cyanomethanol, p-nitrophenol,2,4-dinitrophenol, pentachlorophenol and the like; and active acidamides with saccharin and the like.

As the protective groups for --COO⁻ of the compound of the generalformula (IV), there can be similarly used the groups which werementioned as the protective groups for carboxyl group of the compound ofthe aforesaid general formula (II).

As the protective group for the amino group of the compound of thegeneral formula (V), there can be used the groups which were illustratedas the protective groups for the amino group of the compound of theabove-mentioned general formula (II).

These protective groups can be removed by any conventional manner, suchas hydrolysis, reduction, and the like, depending on the types of theprotective groups used.

As the salts of the compounds of the formulae (IV) and (V), there may besuitably selected the salts which were illustrated as the salts of thecompounds of the general formulae (II) and (III).

The novel compounds represented by the general formula: ##STR9## whereinn and R₂ have the same meanings as defined above, a is 0 or 1, R₃represents hydrogen atom or a protective group for the amino group, Adenotes an anion, and b stands for 0, when R₄ denotes a group --COO⁻,and for 1, when R₄ represents a group --COOR₅ (R₅ being a protectivegroup for the carboxyl group), or a salt thereof, inclusive of thecompounds represented by the general formula (IV) are intermediates forthe compounds represented by the general formula (I) of the presentinvention. These compounds are used for the production of the compoundsof the present invention in accordance with the above-mentioned secondprocess for the production.

Examples of R₃ being the protective group for the amino group in thecompounds represented by said general formula (VI) include thoseemployed usually in this field, for example, substituted orunsubstituted lower alkanoyl groups, such as formyl, acetyl,chloroacetyl, dichloroacetyl, propionyl, phenylacetyl, 2-thienylacetyl,2-furylacetyl, phenoxyacetyl and the like; substituted or unsubstitutedlower alkoxycarbonyl groups such as benzyloxycarbonyl, t-butoxycarbonyl,p-nitrobenzyloxycarbonyl and the like; substituted lower alkyl groupssuch as trityl, p-methoxybenzyl, diphenylmethyl and the like; andsubstituted silyl groups such as trimethylsilyl, t-butyldimethylsilyland the like.

Examples of R₅ being the protective group for the carboxyl group includethose employed usually in this field, for example, substituted orunsubstituted lower alkyl groups such as methyl, ethyl, propyl, t-butyl,2,2,2-trichloroethyl, valeryloxymethyl, pivaloyloxymethyl,p-nitrobenzyl, p-methoxybenzyl, diphenylmethyl and the like; andsubstituted silyl groups such as trimethylsilyl, t-butyldimethylsilyland the like.

Examples of the anion in A include halogen ions such as chloro ion,bromo ion, iodo ion and the like; and inorganic acid ions such assulfuric acid ion, nitric acid ion and the like.

An example of salts of the compounds represented by the general formula(VI) includes inorganic acid salts such as hydrochloride, hydrobromide,hydroiodide, sulfate, carbonate, bicarbonate and the like; organiccarboxylates such as, acetate, maleate, lactate, tartrate,trifluoroacetate and the like; organic sulfonates such asmethanesulfonate, benzenesulfonate, toluenesulfonate and the like; andamino acid salts such as aspartate, glutamate and the like.

The compounds represented by the general formula (VI) can be produced inaccordance with the following process.

A compound represented by the general formula: ##STR10## wherein a andR₃ have the same meanings as defined above, R₆ denotes hydrogen atom ora protective group for the carboxyl group, and Z is a halogen atom or alower alkanoyloxy group, or a salt thereof is reacted with a compoundrepresented by the general formula: ##STR11## wherein n and R₂ have thesame meanings as defined above, or with a salt thereof, followed by, ifnecessary, removal of the protective group and/or reduction of thesulfoxide to obtain the compounds having the aforesaid general formula(VI) or the salts thereof.

In the case wherein Z in the general formula (VII) represents a halogenatom, the above reaction may be carried out in an inert solvent such asacetone, tetrahydrofuran, N,N-dimethylformamide, methylene chloride,chloroform, acetonitrile, and the like at a reaction temperature of -10°C. to 50° C.

In the case wherein Z in the general formula (VII) represents a loweralkanoyloxy group, the above reaction may be carried out in an inertsolvent such as chloroform, methylene chloride, tetrahydrofuran,N,N-dimethylformamide, dioxane, acetone, and the like in the presence ofiodotrimethylsilane at a reaction temperature of -20° C. to 60° C.

The removal of protective groups may be achieved in accordance with aconventional procedure such as hydrolysis, reduction and the like,depending upon the type of the protective group used. Furthermore, thereduction of sulfoxide may be carried out by using a conventionalreduction reagent such as, for example, phosphorus trichloride, and thelike.

An example of Z being the halogen atom in the compound represented bythe general formula (VII) includes chlorine, bromine, or iodine atom.

An example of Z being the lower alkanoyloxy group includes acetyloxy,propionyloxy, and the like.

An example of R₆ being the protective group for carboxyl group includesthose enumerated in the description for R₅. Furthermore, any salt whichdoes not inhibit said reaction may be used as the salts of the compoundrepresented by the general formulae (VII) and (III), and these salts maybe suitably selected from the group consisting of, for example, alkalimetal salts such as sodium, potassium and the like salts; alkaline earthmetal salts such as calcium, magnesium and the like salts; ammoniumsalts; inorganic acid salts such as hydrochloride, sulfate, carbonate,bicarbonate, hydrobromide, hydroiodide, and the like; organiccarboxylates such as acetate, maleate, lactate, tartrate,trifluoroacetate and the like; organic sulfonates such asmethanesulfonate, benzenesulfonate, toluenesulfonate and the like; aminesalts such as trimethylamine, triethylamine, pyridine, procaine,picoline, dicycloheylamine, N,N'-dibenzylethylenediamine,N-methylglucamine, diethanolamine, triethanolamine,tris(hydroxymethylamino)methane and the like salts; and amino acid saltssuch as alginate, aspartate, glutamate and lysine, serine or the likesalt.

The compounds of this invention showed strong antibacterial activitiesagainst both gram-positive and gram-negative bacteria. In addition, inthe case of the following compounds, all of their acute toxicity levels[LD₅₀ (mouse, intravenous injection)] were found to be more than 3 g/kg.

7β-[(Z)-2-(2-Aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-(4-hydroxy-1,4-methylene-1-piperidinio)methyl-3-cephem-4-carboxylate;

7β-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(4-hydroxy-1,4-methylene-1-piperidinio)methyl-3-cephem-4-carboxylate;

7β-[(Z)-2-(2-Aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-(4-hydroxy-1-quinuclidinio)methyl-3-cephem-4-carboxylate;

7β-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(4-hydroxy-1-quinuclidinio)methyl-3-cephem-4-carboxylate;

7β-[(Z)-2-(2-Aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate;and

7β-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate;

When using the compounds of this invention as antibacterial agents,their dosages are 2 to 300 mg/kg/day or preferably 10 to 100 mg/kg/day.

The antibacterial composition may be administered orally in the form ofpowder, granules, capsules, tablets and the like, or parenterally in theform of parenteral solutions, suppositories and the like. Thesecompositions may be prepared in a usual manner, using an effectiveamount of the compound of this invention and pharmaceutically-acceptableexcipients.

By the way, the following nomenclature is used in the present invention.

The present invention will be described in further detail by thefollowing Experiments and Examples.

EXPERIMENT 1 Production of 4-hydroxy-1,4-methylenepiperidine ##STR13##

(1) 1-Benzyl-4-ethoxycarbonyl-4-hydroxypiperidine:

Conc. sulfuric acid (12 ml) was added to an ethanol solution (50 ml) of1-benzyl-4-cyano-4-hydroxypiperidine hydrochloride (10 g) and theresulting mixture was heated at 130° C. for 24 hours in a sealed tube.After concentration of the reaction mixture, ice water was added to theconcentrate. The resulting mixture was added with an aqueous solution ofsodium hydrogencarbonate. After adjusting the thus-obtained soluble topH 7.0, it was extracted with diethyl ether. The extract was washed withbrine and then added with anhydrous sodium sulfate to dry the same. Thesolvent was distilled off to obtain the intended product (9.7 g).

(2) 1-Benzyl-4-hydroxymethyl-4-hydroxypiperidine:

To a diethyl ether suspension (0.5 l) of lithium aluminum hydride (19.4g), a diethyl ether solution (0.5 l) of the compound (44.6 g) obtainedin the above procedure 1) was added dropwise under ice-cooling. Aftercompletion of the dropwise addition, the resulting mixture was stirredfor 1.5 hrs. After addition of ethyl acetate (100 ml) and saturatedaqueous sodium sulfate solution (100 ml) to the reaction mixture, theresulting mixture was filtered through Celite (trade mark). The residuewas washed with tetrahydrofuran. The filtrate and washing were combinedtogether, followed by their concentration under reduced pressure. Theresidue was then purified by alumina column chromatography (eluent:chloroform, 5% methanol-chloroform, and 20% methanol-chloroform) toobtain the desired product (31.5 g).

(3) 1-Benzyl-4-hydroxy-1,4-methylenepiperidine p-toluenesulfonate:

To a pyridine solution (120 ml) of the compound (11.3 g) obtained in theabove procedure 2), p-toluenesulfonyl chloride (10.7 g) was added at-30° C. The resulting mixture was heated to 4° C., at which it wasstirred for 15 hours. The reaction mixture was concentrated underreduced pressure and the thus-obtained residue was dissolved in a smallamount of ice water. After adding 2N aqueous potassium hydroxidesolution (82 ml) to the above-prepared solution, the resulting solutionwas extracted with benzene. Anhydrous potassium carbonate was added tothe extract to dry the same. After refluxing the solution for 9 hours,it was cooled to room temperature. The resulting precipitate wascollected by filtration and then washed with absolute benzene to obtainthe desired product (16.5 g).

(4) 4-Hydroxy-1,4-methylenepiperidine:

Added to a methanol solution (300 ml) of the compound (16.5 g) obtainedin the above procedure (3) was 10% palladium-carbon (50% water content;3.3 g), followed by stirring for 3 hours in a hydrogen atmosphere. Thereaction mixture was filtered and the filtrate was concentrated underreduced pressure. Saturated aqueous potassium carbonate solution wasadded to the residue, followed by extraction with chloroform. Afteradding anhydrous potassium carbonate to the extract to dry the same, thethus-dried extract was concentrated under reduced pressure to obtain thedesired product (4.0 g).

Melting point: 124.0°-124.5° C.

Mass spectrum (M⁺): 113.

NMR spectrum (CDCl₃ : δ): 1.70(4H, m), 2.41(2H, s), 2.75(2H, m),3.16(2H, m).

EXAMPLE 17β-[(Z)-2-(2-Aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-(4-hydroxy-1,4-methylene-1-piperidinio)methyl-3-cephem-4-carboxylate##STR14##

7β-[(Z)-2-(2-Aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid (240 mg) was suspended in methylene chloride (4 ml), followed by anaddition of N-methyl-N-(trimethylsilyl) trifluoroacetamide (330 μl). Theresulting mixture was stirred at room temperature for 30 minutes. Afterice-cooling, iodotrimethylsilane (200 μl) was added to the solution, andthe resulting mixture was stirred at room temperature for 15 minutes.The reaction mixture was concentrated under reduced pressure to obtainthe silylated derivative of7β-[(Z)-2-(2-aminothiazol-4yl)-2-methoxyiminoacetamido]-3-iodomethyl-3-cephem-4-carboxylicacid.

The silylated derivative was dissolved in acetonitrile (3 ml), followedby an addition of tetrahydrofuran (60 μl). The thus-obtained solutionwas added with 4-hydroxy-1,4-methylenepiperidine (72 mg) and theresulting mixture was stirred at room temperature for 2 hours. Methanol(0.3 ml) was then added to the reaction mixture and the resultingmixture was stirred for 15 minutes. The resulting precipitate wascollected by filtration and then washed with acetonitrile. Theprecipitate was dissolved in 30% ethanol. Subsequent to itsconcentration under reduced pressure, the residue was dissolved in a 7:1mixed solvent of acetone and water. The resulting solution was purifiedby silica gel column chromatography (eluent: 9:1 and 7:1 mixed solventof acetone and water) to obtain the desired product (39 mg).

EXAMPLE 27β-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(4-hydroxy-1,4-methylene-1-piperidinio)methyl-3-cephem-4-carboxylate##STR15##

7β-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid (319 mg) was suspended in methylene chloride (4 ml), followed by anaddition of N-methyl-N-(trimethylsilyl)trifluoroacetamide (877 μl). Theresulting mixture was stirred at room temperature for 1 hour. Afterice-cooling, iodotrimethylsilane (268 μl) was added and the resultingmixture was stirred for 15 minutes. The reaction mixture wasconcentrated under reduced pressure to obtain the silylated derivativeof7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-iodomethyl-3-cephem-4-carboxylicacid.

The silylated derivative was dissolved in acetonitrile (3.6 ml). Thethus-obtained solution was added with 4-hydroxy-1,4-methylenepiperizine(71 mg) and the resulting mixture was stirred for 2 hours withice-cooling. Methanol (0.3 ml) was then added to the reaction mixtureand the resulting mixture was stirred for 15 minutes. The resultingprecipitate was collected by filtration and then washed withacetonitrile. The precipitate was dissolved in 30% ethanol. Subsequentto its concentration under reduced pressure, the residue was dissolvedin a 7:1 mixed solvent of acetone and water. The resulting solution waspurified by silica gel column chromatography (eluent: 7:1 and 5:1 mixedsolvent of acetone and water) to obtain the desired product (29 mg).

EXAMPLE 37β-[(Z)-2-(2-Aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-(4-hydroxy-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR16##

7β-[(Z)-2-(2-Aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid (977 mg) was suspended in methylene chloride (16 ml), followed byan addition of N-methyl-N-(trimethylsilyl)trifluoroacetamide (1350 μl).The resulting mixture was stirred at room temperature for 1 hour. Afterice-cooling, iodotrimethylsilane (810 μl) was added and the resultingmixture was stirred for 15 minutes. The reaction mixture wasconcentrated under reduced pressure to obtain the silylated derivativeof7β-[(Z)-2-(2-aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-iodomethyl-3-cephem-4-carboxylicacid.

The silylated derivative was dissolved in acetonitrile (12 ml), followedby an addition of tetrahydrofuran (240 μl). The thus-obtained solutionwas added with 4-hydroxyquinuclidine (300 mg) and the resulting mixturewas stirred at room temperature for 1.5 hours. Methanol (1.2 ml) wasthen added to the reaction mixture and the resulting mixture was stirredfor 15 minutes. The resulting precipitate was collected by filtrationand then washed with acetonitrile. The precipitate was dissolved in 30%ethanol. Subsequent to its concentration under reduced pressure, theresidue was dissolved in a 7:1 mixed solvent of acetone and water. Theresulting solution was purified by silica gel column chromatography(eluent: 7:1 mixed solvent of acetone and water) to obtain the desiredproduct (38 mg).

EXAMPLE 47β-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(4-hydroxy-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR17##

7β-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid (486 mg) was suspended in methylene chloride (9 ml), followed by anaddition of N-methyl-N-(trimethylsilyl)trifluoroacetamide (980 μl). Theresulting mixture was stirred at room temperature for 1 hour. Afterice-cooling, iodotrimethylsilane (410 μl) was added and the resultingmixture was stirred for 15 minutes. The reaction mixture wasconcentrated under reduced pressure to obtain the silylated derivativeof7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-iodomethyl-3-cephem-4-carboxylicacid.

The silylated derivative was dissolved in acetonitrile (6 ml), followedby an addition of tetrahydrofuran (130 μl). The resulting solution wasadded with 4-hydroquinuclidine (150 mg) and the resulting mixture wasstirred at room temperature for 1 hour. Methanol (0.6 ml) was then addedto the reaction mixture and the resulting mixture was stirred for 15minutes. The resultng precipitate was collected by filtration and thenwashed with acetonitrile. The precipitate was dissolved in 30% ethanol.Subsequent to its concentration under reduced pressure, the residue waspurified by silica gel column chromatography (eluent: 9:1 mixed solventof acetone and water) to obtain the desired product (13 mg).

EXAMPLE 57β-[(Z)-2-(2-Aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR18##

7β-[(Z)-2-(2-Aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid (240 mg) was suspended in methylene chloride (4 ml), followed by anaddition of N-methyl-N-(trimethylsilyl)trifluoroacetamide (330 μl). Theresulting mixture was stirred at room temperature for 30 minutes. Afterice-cooling, iodotrimethylsilane (200 μl) was added and the resultingmixture was stirred for 15 minutes. The reaction mixture was thenconcentrated under reduced pressure to obtain the silylated derivativeof7β-[(Z)-2-(2-aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-iodomethyl-3-cephem-4-carboxylicacid.

The silylated derivative was dissolved in acetonitrile (3 ml), followedby an addition of tetrahydrofuran (60 μl). The resulting solution wasadded with 4-carbamoylquinuclidine (98 mg) and the resulting mixture wasstirred at room temperature for 2 hours. Methanol (0.3 ml) was thenadded to the reaction mixture and the resulting mixture was stirred atroom temperature for 15 minutes. The resulting precipitate was collectedby filtration and then washed with acetonitrile. The precipitate wasdissolved in 30% ethanol. Subsequent to its concentration under reducedpressure, the residue was dissolved in a 7:1 mixed solvent of acetoneand water. The thus-obtained solution was purified by silica gel columnchromatography (eluent: 7:1 mixed solvent of acetone and water) toobtain the desired product (53 mg).

EXAMPLE 67β-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR19##

7β-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid (790 mg) was suspended in methylene chloride (10 ml), followed byan addition of N-methyl-N-(trimethylsilyl)trifluoroacetamide (2.1 ml).The resulting mixture was stirred at room temperature for 1 hour. Afterice-cooling, iodotrimethylsilane (660 μl) was added and the resultingmixture was stirred for 15 minutes. The reaction mixture wasconcentrated under reduced pressure to obtain the silylated derivativeof7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-iodomethyl-3-cephem-4-carboxylicacid.

The silylated derivative was dissolved in acetonitrile (9 ml), followedby an addition of 4-carbamoylquinuclidine (240 mg). The resultingmixture was stirred for 1 hour with ice-cooling. Methanol (0.6 ml) wasthen added to the reaction mixture and the resulting mixture was stirredfor 15 minutes. The resulting precipitate was collected by filtrationand then washed with acetonitrile. The precipitate was dissolved in 30%ethanol. Subsequent to its concentration under reduced pressure, theresidue was dissolved in 7:1 mixed solvent of acetone and water. Thesolution was purified by silica gel column chromatography (eluent: 7:1and 5:1 mixed solvent of acetone and water) to obtain the desiredproduct (326 mg).

EXAMPLE 77β-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-carboxymethoxyiminoacetamido]-3-(4-hydroxy-1,4-methylene-1-piperidinio)methyl-3-cephem-4-carboxylate##STR20##

Similar to Examples 1-6, the silylated derivative of7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-carboxymethoxyiminoacetamido]-3-iodomethyl-3-cephem-4-carboxylicacid was obtained from7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-carboxymethoxyiminoacetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid (500 mg), N-methyl-N-(trimethylsilyl)trifluoroacetamide (1.23 ml)and iodotrimethylsilane (780 μl). The silylated derivative was reactedwith 4-hydroxy-1,4-methylenepiperidine (90 mg) to obtain the desiredproduct (113 mg).

EXAMPLE 87β-[(Z)-2-(2-Aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-(4-hydroxymethyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR21##

Similar to Examples 1-6, the silylated derivative of7β-[(Z)-2-(2-aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-iodomethyl-3-cephem-4-carboxylicacid was obtained from7β-[(Z)-2-(2-aminothiazol-4-yl)-2-methoxyiminoacetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid (240 mg), N-methyl-N-(trimethylsilyl)trifluoroacetamide (330 μl)and iodotrimethylsilane (300 μl). The silylated derivative was reactedwith 4-hydroxymethylquinuclidine (89 mg) to obtain the desired product(6 mg).

EXAMPLE 97β-[(Z)-2-(2-Aminothiazol-4-yl)-2-propargyloxyiminoacetamido]-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR22##

Similar to Examples 1-6, the silylated derivative of7β-[(Z)-2-(2-aminothiazol-4-yl)-2-propargyloxyiminoacetamido]-3-iodomethyl-3-cephem-4-carboxylicacid was obtained from7β-[(Z)-2-(2-aminothiazol-4-yl)-2-propargyloxyiminoacetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid (290 mg), N-methyl-N-(trimethylsilyl)trifluoroacetamide (380 μl)and iodotrimethylsilane (230 μl). The silylated derivative was reactedwith 4-carbamoylquinuclidine (112 mg) to obtain the desired product (10mg).

EXAMPLE 107β-[(Z)-2-(2-Aminothiazol-4-yl)-2-carbamoylmethoxyiminoacetamido]-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR23##

Similar to Examples 1-6, the silylated derivative of7β-[(Z)-2-(2-aminothiazol-4-yl)-2-carbamoylmethoxyiminoacetamido]-3-iodomethyl-3-cephem-4-carboxylicacid was obtained from7β-[(Z)-2-(2-aminothiazol-4-yl)-2-carbamoylmethoxyiminoacetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid (110 mg), N-methyl-N-(trimethylsilyl)trifluoroacetamide (140 μl)and iodotrimethylsilane (180 μl). The silylated derivative was reactedwith 4-carbamoylquinuclidine (41 mg) to obtain the desired product (5mg).

EXAMPLE 117β-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-ethoxyiminoacetamido]-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR24##

Similar to Examples 1-6, the silylated derivative of7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-ethoxyiminoacetamido]-3-iodomethyl-3-cephem-4-carboxylicacid was obtained from7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-ethoxyiminoacetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid (300 mg), N-methyl-N-(trimethylsilyl)trifluoroacetamide (600 μl)and iodotrimethylsilane (500 μl). The silylated derivative was reactedwith 4-carbamoylquinuclidine (118 mg) to obtain the desired product (62mg).

EXAMPLE 127β-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-(1-carboxy-1-methylethoxy)iminoacetamido]-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR25##

Similar to Examples 1-6, the silylated derivative of7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(1-carboxy-1-methylethoxy)iminoacetamido]-3-iodomethyl-3-cephem-4-carboxylicacid was obtained from7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(1-carboxy-1-methoxyethoxy)iminoacetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid (530 mg), N-methyl-N-(trimethylsilyl)trifluoroacetamide (820 μl)and iodotrimethylsilane (390 μl). The silylated derivative was reactedwith 4-carbamoylquinuclidine (186 mg) to obtain the desired product (100mg).

EXAMPLE 137β-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-carboxymethoxyiminoacetamido]-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR26##

Similar to Examples 1-6, the silylated derivative of7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-carboxymethoxyiminoacetamido]-3-iodomethyl-3-cephem-4-carboxylicacid was obtained from7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-carboxymethoxyiminoacetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid (500 mg), N-methyl-N-(trimethylsilyl)trifluoroacetamide (1.23 μl)and iodotrimethylsilane (780 μl). The silylated derivative was reactedwith 4-carbamoylquinuclidine (142 mg) to obtain the desired product (36mg).

EXAMPLE 147β-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[4-hydroxymethyl-1-quinuclidinio]methyl-3-cephem-4-carboxylate##STR27##

Similar to Examples 1-6, the silylated derivative of7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-iodomethyl-3-cephem-4-carboxylicacid was obtained from7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-acetoxymethyl-3-cephem-4-carboxylicacid (460 mg), N-methyl-N-(trimethylsilyl)trifluoroacetamide (640 μl),and iodotrimethylsilane (390 μl). The silylated derivative was reactedwith 4-hydroxylmethylquinuclidine (142 mg) to obtain the desired product(8 mg).

EXAMPLE 157β-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(4-methyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR28##

p-Methoxybenzyl7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-iodomethyl-3-cephem-4-carboxylate(700 mg) was dissolved in a mixed solution of ethyl acetate (50 ml) andmethanol (1 ml). After the whole was ice-cooled, a solution of ethylacetate (2.8 ml) of 4-methyl-quinuclidine (114 mg) was added thereto,and the mixture was stirred for 15 minutes. The resulting precipitatewas recovered by filtration, followed by wasing with ethyl acetate toobtain p-methoxybenzyl 7β-[(Z)-2-(5-amino1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(4-methyl-1-quinuclidinio)methyl-3-cephem-4-carboxylateiodide (770 mg).

This compound (770 mg) was suspended in methylene chloride (8 ml). Afterice-cooling, anisole (510 μl) and trifluoroacetic acid (730 μl) wereadded thereto. The mixture was stirred for 4 hours, followed by stirringfor an additional 2.5 hours at room temperature. The resulting reactionsolution was dropped in diisopropyl ether (30 ml), and the resultingprecipitate was collected by filtration. The precipitate dissolved inwater (5 ml). The solution was adjusted to pH of 5.0 by the addition ofsodium hydrogencarbonate. The mixture was subjected to a reversed phasesilica gel column chromatography (eluent: water→5% methanol solution )for purification, to obtain the desired product (27 mg).

EXAMPLE 167β[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-cyclopropylmethoxyiminoacetamido]-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR29##

p-Methoxybenzyl7β-[(Z)-2-(5-t-butoxycarboxamido-1,2,4-thiadiazol-3-yl)-2-cyclopropylmethoxyiminoacetamido]-3-iodomethyl-3-cephem-4-carboxylate(450 mg) was dissolved in ethyl acetate (30 ml). After ice-cooling,there was added a mixted solution of methanol (1 ml) and ethyl acetate(5 ml) of 4-carbamoylquinuclidine (80 mg), and the whole was stirred for30 minutes. The formed precipitate was collected by filtration, followedby washing with ethyl acetate to obtain p-methoxybenzyl7β-[(Z)-2-(5-t-butoxycarboxamido-1,2,4-thiadiazol-3-yl)-2-cyclopropylmethoxyiminoacetamido]-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylateiodide (290 mg).

This compound (290 mg) was dissolved in formic acid (6 ml), and theresulting solution was stirred for a day at room temperature. To thereaction solution were added acetone (10 ml), diisopropyl ether (30 ml)and n-hexane (50 ml). The formed precipitate was filtered off, and thefiltrate was concentrated under a reduced pressure. The residue wasdissolved in a water-methanol solution (10 ml), and the solution wasadjusted to pH of 5.5 by the addition of sodium hydrogencarbonate. Theresulting solution was concentrated, followed by purifying throughreversed phase silica gel column chromatography (eluent: water→5%methanol solution) to obtain the desired product (19 mg).

EXAMPLE 177β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-cyclopropylmethoxyiminoacetamido]-3-(4-hydroxymethyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR30##

Similar to Example 16, p-methoxybenzyl7β-[(Z)-2-(5-t-butoxycarboxamido-1,2,4-thiadiazol-3-yl)-2-cyclopropylmethoxyiminoacetamido]-3-iodomethyl-3-cephem-4-carboxylate(450 mg) was reacted with 4-hydroxymethylquinuclidine (73 mg) to obtainp-methoxybenzyl7β-[(Z)-2-(5-t-butoxycarboxamido-1,2,4-thiadiazol-3-yl)-2-cyclopropylmethoxyiminoacetamido]-3-(4-hydroxymethyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate(310 mg), followed by removing a protective group by means of formicacid to obtain the desired product (23 mg).

                                      TABLE 1                                     __________________________________________________________________________    Physical Data                                                                 Example                                                                            IR absorption                                                            No.  spectrum (cm.sup.-1, Nujol)                                                               NMR spectrum (δ, D.sub.2 O)                            __________________________________________________________________________     1   1765        2.30(4H,m), 3.20-4.40(m) 4.08(3H,s),                                          5.43(1H,d,J = 6 Hz), 5.94(1H,d,J = 6 Hz), 7.09(1H,s).         2   1775        2.32(4H,m), 3.30-4.00(m), 4.18(3H,s),                                         5.43(1H,d,J = 6 Hz), 5.98(1H,d,J = 6 Hz)                      3   1765        2.20(6H,m), 3.40-4.00(m), 4.10(3H,s),                                         5.44(1H,d,J = 6 Hz), 5.96(1H,d,J = 6 Hz), 7.10(1H,s).         4   1765        2.20(6H,m), 3.30-4.00(m), 4.18(3H,s),                                         5.43(1H,d,J = 6 Hz), 5.97(1H,d,J = 6 Hz).                     5   1770        2.30(6H,m), 3.30-4.00(m), 4.10(3H,s),                                         5.45(1H,d,J = 6 Hz), 5.97(1H,d,J = 6 Hz), 7.12(1H,s).         6   1775        2.30(6H,m), 3.15-4.00(m), 4.16(3H,s),                                         5.43(1H,d,J = 6 Hz), 5.97(1H,d,J = 6 Hz).                     7   1760        2.30(4H,m), 3.00-4.30(m), 4.77(2H,s),                                         5.42(1H,d,J = 6 Hz), 5.99(1H,d,J = 6 Hz).                     8   1770        1.96(6H,m), 3.20-4.00(m), 4.12(3H,s),                                         5.47(1H,d,J = 6 Hz), 5.97(1H,d,J = 6 Hz), 7.15(1H,s).         9   1765        2.24(6H,m), 3.20-4.20(m), 5.47(1H,d,J = 6 Hz),                                5.99(1H,d,J = 6 Hz), 7.19(1H,s).                             10   1770        2.30(6H,m), 3.30-4.20(m), 5.48(1H,d,J = 6 Hz),                                6.00(1H,d,J = 6 Hz), 7.22(1H,s).                             11   1770        1.45(3H,t,J = 8 Hz), 2.30(6H,m), 3.20-4.20(m),                                4.46(2H,q,J = 8 Hz), 5.46(1H,d,J = 6 Hz), 6.00(1H,d,J =                       6 Hz).                                                       12   1770        1.65(6H,s), 2.25(6H,m), 3.30-4.20(m),                                         5.47(1H,d,J = 6 Hz), 6.01(1H,d,J = 6 Hz).                    13   1765        2.30(6H,m), 3.20-4.30(m),                                                     5.44(1H,d,J = 5 Hz), 6.00(1H,d,J = 5 Hz).                    14   1770        1.96(6H,m), 3.20-4.30(m), 4.19(3H,s)                                          5.44(1H,d,J = 6 Hz), 5.99(1H,d,J = 6 Hz).                    15   1765        1.14(3H,s), 1.94(6H,m), 3.30-4.20(m), 4.20(3H,s),                             5.47(1H,d,J = 6 Hz), 6.01(1H,d,J = 6 Hz)                     16   1770        0.30-0.90(4H,m), 1.37(1H,m), 2.30(6H,m), 3.20-4.20(m),                        4.25(2H,d,J = 8 Hz), 5.47(1H,d,J = 5 Hz), 6.02(1H,d,J =                       5 Hz).                                                       17   1770        0.30-0.90(4H,m), 1.37(1H,m), 2.04(6H,m), 3.20-4.10(m)                         4.25(2H,d,J = 8 Hz), 5.48(1H,d,J = 6 Hz), 6.03(1H,d,J =                       6 Hz).                                                       __________________________________________________________________________

EXPERIMENT 27β-Formamido-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR31##

7β-Formamido-3-acetoxymethyl-3-cephem-4-carboxylic acid (1.2 g) wassuspended in methylene chloride (12 ml), and thenN-methyl-N-(trimethylsilyl)trifluoroacetamide (815 μl) was added theretoand stirred for 30 minutes. After cooling the mixture with ice,iodotrimethylsilane (1.25 ml) was added thereto and stirred for 5minutes, thereafter the temperature of the mixture was returned to roomtemperature, and the mixture was stirred for another 15 minutes. Solventwas distilled away under reduced pressure from the resulting solution,and the residue was dissolved in acetonitrile (12 ml).4-Carbamoylquinuclidine (616 mg) was added to the solution underice-cooling and stirred for 1 hour. To the reaction solution was addedmethanol (3 ml) and further diethyl ether (300 ml), and the resultingprecipitate was filtered.

The precipitate was purified by means of silica gel columnchromatography [eluent: acetone-water (7:1) and (5:1)] to obtain thedesired product (140 mg).

EXPERIMENT 37β-Tritylamino-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR32##

7β-Tritylamino-3-acetoxymethyl-3-cephem-4-carboxylic acid (2.4 g) wasdissolved in methylene chloride (24 ml), and thenN-methyl-N-(trimethylsilyl)trifluoroacetamide (960 μl) was added theretoand stirred for 30 minutes. After cooling the mixture with ice,iodotrimethylsilane (720 μl) was added thereto and stirred for 5minutes, thereafter the temperature of the mixture was returned to roomtemperature, and the mixture was stirred for another 15 minutes. Solventwas distilled away under reduced pressure from the resulting solution,and the residue was dissolved in acetonitrile (12 ml).4-Carbamoylquinuclidine (756 mg) was added to the solution underice-cooling and stirred for 1 hour. To the reaction solution was addedmethanol (3.2 ml) and then diethyl ether (240 ml), and the resultingprecipitate was filtered.

The precipitate was purified by means of silica gel columnchromatography [eluent: acetone-water (7:1), (5:1), and (3:1)] to obtainthe desired product (207 mg).

EXPERIMENT 47β-(2-Thienylacetamido)-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR33##

7β-(2-Thienylacetamido)-3-acetoxymethyl-3-cephem-4-carboxylic acid (6.0g) was suspended in methylene chloride (60 ml), and thenN-methyl-N-(trimethylsilyl)-trifluoroacetamide (3.08 ml) was addedthereto and stirred for 30 minutes. After cooling the mixture with ice,iodotrimethylsilane (4.73 ml) was added thereto and stirred for 5minutes, thereafter the mixture was further stirred at room temperaturefor another 15 minutes. The resulting solution was concentrated underreduced pressure, and the residue was dissolved in acetonitrile (60 ml).After ice-cooling the resulting solution, 4-carbamoylquinuclidine (2.3g) was added to the solution and stirred for 1 hour. To the reactionsolution was added methanol (6 ml), and then diethyl ether (600 ml) wasdropoed thereto. After stirring the mixture for 1 hour, the resultingprecipitate was filtered.

The precipitate was purified by means of silica gel columnchromatography [eluent: acetone-water (7:1) and and (5:1)] to obtain thedesired product (700 mg).

EXPERIMENT 57β-Amino-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylatehydrochloride ##STR34##

The compound (130 mg) prepared in Experiment 2 was suspended in methanol(5 ml), and concentrated hydrochloric acid (0.52 ml) was added to thesuspension at room temperature and stirred for 4 hours. The reactionmixture was concentrated under reduced pressure and crystallized bymeans of ethyl ether-methanol, whereby the desired product (115 mg) wasobtained.

EXPERIMENT 67β-Amino-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylatehydrochloride ##STR35##

The compound (100 mg) prepared in Experiment 3 was suspended in 50%formic acid (5 ml), and stirred at room temperature for 3.5 hours. Tothe suspension was added water (20 ml) and insoluble matter was filteredoff, and the filtrate was concentrated under reduced pressure. Theresidue was dissolved in 1N hydrochloric acid (1 ml), and propanol (5ml) and diethyl ether (10 ml) was added thereto. The precipitate wasfiltered, washed with n-hexane, and then dried, whereby the desiredproduct (45 mg) was obtained.

EXPERIMENT 77β-Amino-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylatehydrochloride ##STR36##

The compound (600 mg) prepared in Experiment 4 was suspended inmethylene chloride (30 ml), N,N-dimethylaniline (1.24 ml) andchlorotrimethylsilane (465 μl) were added thereto, and mixture wasstirred at 30° C. for 3 hours. Then, the reaction mixture was cooled to-25° C., thereafter phosphorus pentachloride (1.27 g) was added thereto,and the mixture was stirred for 1 hour. To the solution was added anice-cooled solution of 1,3-butanediol (1.3 ml) in methylene chloride (25ml), and the mixture was stirred at the same temperature for 10 minutes.The reaction mixture was warmed to 0° C. and stirred further for 40minutes, and then the resulting precipitate was filtered. Theprecipitate was dissolved in methanol (7 ml), and the insoluble matterwas filtered off. Thereafter methylene chloride (20 ml) and diethylether (20 ml) were added to the filtrate, so that the precipitate wasfiltered to obtain the desired product (30 mg).

EXPERIMENT 8 t-Butyl7β-amino-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate1-oxide bromide ##STR37##

t-Butyl 7β-amino-3-bromomethyl-3-cephem-4-carboxylate 1-oxidehydrobromide (600 mg) was dissolved in N,N-dimethylformamide (6 ml), and4-carbamoylquinuclidine (456 mg) was added thereto, and the mixture wasstirred in argon gas stream at room temperature for 14 hours. To thereaction solution was added diethyl ether (120 ml), the resultingprecipitate was filtered and washed with n-hexane, whereby the desiredproduct (580 mg) was obtained.

EXPERIMENT 9 t-Butyl7β-amino-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylatebromide hydrochloride ##STR38##

The compound (570 mg) prepared in Experiment 8 was dissolved inN,N-dimethylformamide (10 ml), phosphorus trichloride (500 μl) was addedto the solution at -25° C. and stirred for 30 minutes. To the reactionsolution was added diethyl ether (50 ml), so that the separated oil wastaken out and washed with diethyl ether, thereafter the resulting solidwas dried under reduced pressure to obtain the desired product (164 mg).

EXPERIMENT 107β-Amino-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR39##

To the compound (150 mg) prepared in Experiment 9 was added formic acid(1.5 ml) and concentrated hydrochloric acid (0.15 ml) under ice-cooledcondition, and the mixture was stirred for 4 hours, thereafterconcentrated under reduced pressure. The residue was dissolved in icewater (5 ml) and neutralized with sodium bicarbonate. The resultingproduct was purified with reversed phase silica gel columnchromatography (eluent: water) to obtain the desired product (60 mg).

EXPERIMENT 11 p-Methoxybenzyl7β-phenylacetamido-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylateiodide ##STR40##

p-Methoxybenzyl 7β-phenylacetamido-3-chloromethyl-3-cephem-4-carboxylate(980 mg) was suspended in acetone (20 ml), and sodium iodide (362 mg)was added to the suspension and stirred at room temperature for 1 hour.To the resulting suspension was added 4-carbamoylquinuclidine (313 mg)under ice-cooled condition, and the mixture was stirred for 2 hours. Thereaction mixture was filtered, and diethyl ether (70 ml) was added tothe filtrate. The deposited solid was filtered to obtain the desiredproduct (500 mg).

                                      TABLE 2                                     __________________________________________________________________________    List of Physical Properties                                                   Experiment                                                                          Infrared Absorption Spectrum                                            No.   (cm.sup.-1, Nujol)                                                                              NMR SPECTRUM (δ)                                __________________________________________________________________________     2    1770              D.sub.2 O: 2.30(6H,m), 3.2-5.0(m), 5.39(1H,d,J =                              6 Hz), 5.89(1H,d,J = 6 Hz), 8.35(1H,s)                 3    1760              D.sub.2 O--Acetone-d.sub.6 : 2.10(6H,m),                                      3.0-4.0(m), 4.60(1H,d,J = 6 Hz),                                              4.70(1H,d,J = 6 Hz), 4.85(1H,d,J = 14 Hz),                                    7.10-7.60(15H,m)                                       4    1775              D.sub.2 O--Acetone-d.sub.6 : 2.15(6H,m),                                      3.0-4.4(m), 3.80(2H,s), 4.81(1H,d,J = 13 Hz),                                 5.16(1H,d,J = 6 Hz), 5.63(1H,d,J = 6 Hz),                                     6.80-6.95(2H,m), 7.20(1H,m)                           5,6,7 1780              D.sub.2 O: 2.30(6H,m), 3.3-4.9(m), 5.31(1H,d,J =                              6 Hz), 5.53(1H,d,J = 6 Hz)                             8    1780              D.sub.2 O--Acentone-d.sub.6 : 2.15(6H,m),                                     3.2-4.4(m), 4.95(2H,br.s), 1.46(9H,s),                                        4.78(1H,d,J = 15 Hz)                                   9    1780              D.sub.2 O: 1.60(9H,s), 2.40(6H,m), 3.2-4.4(m),                                4.71(1H,d,J = 15 Hz),                                                         5.45(1H,d,J = 6 Hz), 5.74(1H,d,J = 6 Hz)              10    1760              D.sub.2 O: 2.50(6H,m), 3.2-5.0(m), 5.32(1H,d,J =                              6 Hz), 5.41(1H,d,J = 6 Hz)                            11    1780              D.sub.2 O--Acetone-d.sub.6 : 2.16(m), 3.1-3.9(m),                             3.59(2H,s), 3.75(3H,s),                                                       4.22(1H,d,J = 14 Hz), 4.57(1H,d,J = 14 Hz),                                   5.24(3H,m), 5.73(1H,8,J = 5 Hz,10 Hz),                                        6.92(2H,d,J = 10 Hz), 7.21(5H,s), 7.39(2H,d,J =                               10 Hz), 8.69(1H,d,J = 10 Hz)                          __________________________________________________________________________

EXPERIMENT 12 p-Methoxybenzyl7β-formamido-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylateiodide ##STR41##

To an ethyl acetate solution (465 ml) of p-methoxybenzyl7β-formamido-3-iodomethyl-3-cephem-4-carboxylate (9.3 g) there was addeddropwise over 1 hour under stirring while ice-cooling a mixed solution(176 ml) of methanol and ethyl acetate (1:4 v/v) containing 4-carbamoylquinuclidine (2.94 g). After stirring for 30 minutes, the resultingprecipitate was collected by filtration, followed by washing with ethylacetate and then diisopropyl ether to obtain the desired product (12.0g).

Infrared absorption spectrum (cm⁻¹, Nujol): 1780

NMR spectrum (δ, D₂ O-acetone-d₆): 1.9-2.4(m), 3.50(8H, m), 3.70(3H, s),4.16(1H, d, J=15 Hz), 4.54(1H, d, J=15 Hz), 5.24(2H, m), 5.78(1H, d, J=5Hz), 6.87(2H, d, J=10 Hz), 7.33(2H, d, J=10 Hz).

EXPERIMENT 137β-Formamido-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR42##

The compound (11.8 g) obtained in the preceding Experiment 12 wasdissolved in ice-cooled formic acid (50 ml), followed by stirring for 10hours at room temperature. The reaction solution was subjected tofiltration, and the filtrate was added dropwise into acetone (100 ml).To the resulting solution was added dropwise further diisopropyl ether(200 ml). The formed precipitate was collected by filtration, followedby washing with acetone. The precipitate was dissolved in dimethylformamide (30 ml). The solution was added dropwise in acetone (150 ml).The formed precipitate was collected by filtration, followed by washingwith acetone and diisopropyl ether respectively. Thereafter, the solidwas dried under a reduced pressure to obtain the desired product (6.66g).

Infrared absorption spectrum (cm⁻¹, Nujol): 1770

NMR spectrum (δ, D₂ O): 2.30(6H, m), 3.2-5.0(m), 5.39(1H, d, J=6 Hz),5.89(1H, d, J=6 Hz), 8.35(1H, s).

EXAMPLE 187β-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR43##

A mixture consisting of2-(5-amino-1,2,4-thiadizaol-3-yl)-(Z)-2-methoxyiminoacetic acid (46 mg),1-hydroxy-1H-benzotriazole hydrate (35 mg),N,N'-dicyclohexylcarbodiimide (52 mg), and N,N-dimethylformamide (1 ml)was stirred at room temperature for 3 hours, then the mixture wasfiltered, and the filtrate was cooled to 0° C. The resulting solutionwas added to an ice-cooled solution of7β-amino-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylicacid hydrochloride (100 mg), N,N-dimethylformamide (2 ml), andN,N-dimethylaniline (72 μl). After stirring the mixed solution at roomtemperature for 14 hours, the reaction mixture was filtered, and thefiltrate was dropped into diethyl ether (100 ml), while stirring themixture. The deposited precipitate was filtered out and washed withdiethyl ether. To the washed precipitate was added water (10 ml), andinsoluble matter was filtered off. The resulting filtrate was purifiedwith reversed phase silica gel column chromatography to obtain thedesired product (3 mg).

Infrared absorption spectrum (cm⁻¹, Nujol): 1775

NMR spectrum (δ, D₂ O): 2.30(6H, m), 3.1-4.0(m), 4.16(3H, s), 5.43(1H,d, J=6 Hz), 5.97(1H, d, J=6 Hz)

EXAMPLE 197β-[(Z)-2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylate##STR44##

7β-Amino-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylicacid hydrochloride (2 g) was dissolved in acetonitrile-water (1:1) mixedsolution (40 ml), and triethylamine (2.08 ml) was added to the solution.The resulting solution was cooled with ice and2-(5-amino-1,2,4-thiadiazol-3-yl)-(Z)-2-methoxyiminoacetylchloride (2.55g) was added thereto, and the mixture was stirred for 50 minutes. Thereaction solution was added to ethanol (200 ml), the deposited solid wasfiltered, and the solid was washed with ethanol and isopropyl ether,whereby the desired product (450 mg) was obtained.

Infrared absorption spectrum as well as NMR spectrum of the productcoincided with those of Example 18.

The compounds of the following Examples 20-28 were synthesized in thesame manner as those of Examples 18 and 19.

                                      TABLE 3                                     __________________________________________________________________________    Physical data                                                                                         Infrared Absorption                                                           Spectrum                                              Example                                                                            n Y  R.sub.1 R.sub.2                                                                             (cm.sup.-1 Nujol)                                                                       NMR SPECTRUM (δ,                      __________________________________________________________________________                                      D.sub.2 O)                                  20   2 CH --CH.sub.3                                                                            --CONH.sub.2                                                                        1770      2.30(6H,m), 3.30-4.00(m), 4.10(3H,s),                                         5.45(1H,d,J = 6 Hz), 5.97(1H,d,J = 6                                          Hz), 7.12(1H,s)                             21   2 N  --CH.sub.3                                                                            --OH  1765      2.20(6H,m), 3.30-4.00(m), 4.18(3H,s),                                         5.43(1H,d,J = 6 Hz), 5.97(1H,d,J = 6                                          Hz)                                         22   2 CH --CH.sub.3                                                                            --CH.sub.2 OH                                                                       1770      1.96(6H,m), 3.20-4.00(m), 4.12(3H,s),                                         5.47(1H,d,J = 6 Hz), 5.97(1H,d,J = 6                                          Hz), 7.15(1H,s)                             23   2 CH --CH.sub.2 C.tbd.CH                                                                   --CONH.sub.2                                                                        1765      2.24(6H,m), 3.20-4.20(m), 5.47(1H,d,J =                                       6 Hz),                                                                        5.99(1H,d,J = 6 Hz), 7.19(1H,s)             24   2 CH --CH.sub.2 CONH.sub.2                                                                 --CONH.sub.2                                                                        1770      2.30(6H,m), 3.30-4.20(m), 5.48(1H,d,J =                                       6 Hz),                                                                        6.00(1H,d,J = 6 Hz), 7.22(1H,s)             25   2 N  --C.sub.2 H.sub.5                                                                     --CONH.sub.2                                                                        1770      1.45(3H,t,J = 8 Hz), 2.30(6H,m),                                              3.20-4.20(m),                                                                 4.46(2H,q,J = 8 Hz), 5.46(1H,d,J = 6                                          Hz), 6.00(1H,d,J = 6 Hz)                    26   2 N  --CH.sub.2 COOH                                                                       --CONH.sub.2                                                                        1765      2.30(6H,m), 3.20-4.30(m), 5.44(1H,d,J =                                       5 Hz),                                                                        6.00(1H,d,J = 5 Hz)                         27   1 N  --CH.sub.3                                                                            --OH  1775      2.32(4H,m), 3.30-4.00(m), 4.18(3H,s),                                         5.43(1H,d,J = 6 Hz), 5.98(1H,d,J = 6                                          Hz)                                         28   1 CH --CH.sub.3                                                                            --OH  1765      2.30(4H,m), 3.20-4.40(m), 4.08(3H,s),                                         5.43(1H,d,J = 6 Hz), 5.94(1H,d,J = 6                                          Hz), 7.09(1H,s)                             __________________________________________________________________________

                                      TABLE 4                                     __________________________________________________________________________    Effects of the Invention                                                      Antibacterial Activities                                                      Test bacterium                                                                MIC (μg/ml)                                                                Sample                                                                               Staphylococcus                                                                       Staphylococcus                                                                        Escherichia                                                                         Pseudomonas                                                                          Serratia                                                                            Pseudomonas                                                                         Citrobacter                    compound                                                                            aureus  aureus  coli  aeruginosa                                                                           marcescens                                                                          maltophilia                                                                         freundii                       Example                                                                             209-P   E31106* NIHJ  EP-01  ES-75 E04004                                                                              EC-34*                         __________________________________________________________________________    1     0.8     100     0.05  1.56   0.1   25    0.1                            2     0.8     50      0.1   0.8    0.2   12.5  0.1                            3     0.8     100     0.05  1.56   0.1   6.25  0.1                            4     1.56    25      0.1   0.8    0.2   6.25  0.1                            5     1.56    100     0.05  1.56   0.05  12.5  0.1                            6     3.13    25      0.1   0.4    0.2   3.13  0.1                            14    1.56    50      0.1   0.8    0.2   3.13  0.1                            15    0.8     50      0.05  0.8    0.2   1.56  0.05                           __________________________________________________________________________     *Lactamase producing bacteria.                                           

What is claimed is:
 1. A cephem compound represented by the formula:##STR45## wherein n represents 1 or 2, Y represents a nitrogen atom, R₁represents lower alkyl, lower alkenyl, lower alkynyl,carboxyl-substituted lower alkyl, or carbamoyl-substituted lower alkyl,and R₂ represents hydroxyl, hydroxy-substituted lower alkyl, orcarbamoyl, or a pharmaceutically acceptable salt thereof.
 2. Thecompound as claimed in claim 1, wherein R₁ is a lower alkyl group. 3.The compound as claimed in claim 1, wherein R₁ stands for methyl orethyl and R₂ stands for hydroxyl group or carbamoyl.
 4. The compound asclaimed in claim 1, which is7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(4-hydroxy-1,4-methylene-1-piperidinio)methyl-3-cephem-4-carboxylaterepresented by the following formula: ##STR46## or a pharmaceuticallyacceptable salt thereof.
 5. The compound as claimed in claim 1, which is7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(4-hydroxy-1-quinuclidinio)methyl-3-cephem-4-carboxylaterepresented by the following formula: ##STR47## or a pharmaceuticallyacceptable salt thereof.
 6. The compound as claimed in claim 1, which is7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(4-carbamoyl-1-quinuclidinio)methyl-3-cephem-4-carboxylaterepresented by the following formula: ##STR48## or a pharmaceuticallyacceptable salt thereof.
 7. The free acid form of the compound asclaimed in claim
 6. 8. The pharmaceutically acceptable salt form of thecompound as claimed in claim
 6. 9. An antibacterial compositioncomprising a compound represented by the formula: ##STR49## wherein nrepresents 1 or 2, Y represents a nitrogen atom, R₁ represents loweralkyl, lower alkenyl, lower alkynyl, carboxyl-substituted lower alkyl,or carbamoyl-substituted lower alkyl, and R₂ represents hydroxyl,hydroxy-substituted lower alkyl, or carbamoyl, or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carriertherefor.